Summary

A dominant molecular explanation for neural induction is the `default
model', which proposes that the ectoderm is pre-programmed towards a neural
fate, but is normally inhibited by endogenous BMPs. Although there is strong
evidence favouring this in Xenopus, data from other organisms suggest
more complexity, including an involvement of FGF and modulation of Wnt.
However, it is generally believed that these additional signals also act by
inhibiting BMPs. We have investigated whether BMP inhibition is necessary
and/or sufficient for neural induction. In the chick, misexpression of BMP4 in
the prospective neural plate inhibits the expression of definitive neural
markers (Sox2 and late Sox3), but does not affect the early
expression of Sox3, suggesting that BMP inhibition is required only
as a late step during neural induction. Inhibition of BMP signalling by the
potent antagonist Smad6, either alone or together with a dominant-negative BMP
receptor, Chordin and/or Noggin in competent epiblast is not sufficient to
induce expression of Sox2 directly, even in combination with FGF2,
FGF3, FGF4 or FGF8 and/or antagonists of Wnt signalling. These results
strongly suggest that BMP inhibition is not sufficient for neural induction in
the chick embryo. To test this in Xenopus, Smad6 mRNA was
injected into the A4 blastomere (which reliably contributes to epidermis but
not to neural plate or its border) at the 32-cell stage: expression of neural
markers (Sox3 and NCAM) is not induced. We propose that
neural induction involves additional signalling events that remain to be
identified.

Here, we have re-evaluated the participation of BMP and BMP antagonism
during neural induction. We chose to manipulate the BMP pathway
intracellularly in a cell-autonomous way, taking advantage of Smad6, the
inhibitory Smad (Imamura et al.,
1997; Hata et al.,
1998). BMP signalling starts with the binding of extracellular BMP
dimer to BMP receptor type II (BMPRII), which is then able to recognize BMP
receptor type I (BMPRI) forming a tertiary complex, where BMPRII activates
BMPRI by phosphorylation. In turn, active BMPRI recruits Smad1/Smad5/Smad8
proteins to the membrane and activates them by phosphorylation, which allows
them to bind to Smad4; the complex then translocates to the nucleus to
regulate transcription (von Bubnoff and
Cho, 2001). A more divergent group of Smad proteins has been
described: the inhibitory Smads (Smad6/Smad7/Smad10). Although Smad7 inhibits
both activin/nodal-related and BMP/TGFβ signalling, Smad6 is a potent and
specific antagonist of the BMP pathway. It acts by actively associating with
and blocking BMPRI, as well as by competing with Smad4 to bind phosphorylated
Smad1/5/8. In addition, Smad6 can inhibit an alternative BMP intracellular
signalling pathway involving TCF3/Lef1/β-catenin
(von Bubnoff and Cho,
2001).

We show that although forced BMP expression in the neural plate inhibits
the expression of the definitive neural marker Sox2, it does not
affect expression of the earlier marker Sox3. Moreover, BMP
inhibition is not sufficient for neural induction, either in competent chick
epiblast or in the prospective ventral epidermis of Xenopus. These
results suggest that BMP inhibition is a relatively late step in a molecular
cascade leading to the acquisition of neural identity. We tested the proposal
of Wilson and Edlund (Wilson and Edlund,
2001) for the chick embryo by investigating whether combinations
of FGF, BMP inhibition and Wnt inhibition might suffice; we find that no
combination of these can induce neural tissue in vivo.

Materials and methods

Chick experiments

Fertilized hens' eggs (Brown Bovan Gold; Henry Stewart and Company) were
incubated at 38°C to the desired stages. Electroporations were performed
as described (Sheng et al.,
2003). The coding region of chicken Smad6 (a kind gift
from P Szendro and G Eichele) (Yamada et
al., 1999), chick Chordin
(Streit et al., 1998),
Xenopus truncated BMP receptor
(Suzuki et al., 1994),
Xenopus BMP4 (a kind gift from K. Howarth and P. Sharpe) and chick
Cerberus (Zhu et al., 1999)
were cloned into pCAβ-IRES-GFP. All DNA solutions for
electroporation were at 1 μg/μl except cSmad6, which was used at 2μ
g/μl. FGFs were delivered bound to heparin beads (prepared as
described) (Streit et al.,
2000). For each FGF, a concentration that did not induce
brachyury was determined. FGF2 (Invitrogen 13256-029) induces
brachyury at 50 μg/ml (5/5) and 2.5 μg/ml (12/12), but not at
0.5 μg/ml (0/7), and this concentration was therefore used for further
analysis. FGF3 (R&D Systems 1206-F3) was used at 50 μg/ml. FGF4
(R&D Systems 235-F4) induces brachyury at 2.5 μg/ml (9/9) and
0.5 μg/ml (9/9), but not at 0.05 μg/ml (0/13), and this concentration
was used for experiments. Different batches of FGF8b (50 μg/ml) produced
different results when used at the same concentration. In the present set of
experiments, FGF8b obtained from Sigma (F6926) was considerably more potent in
inducing Sox3 (and also induced mesoderm at the area pellucida/area
opaca border, as marked by brachyury expression) than that obtained
from R&D Systems (423-F8), which never induced brachyury. The
latter was therefore used in our experiments at this concentration. Other
secreted proteins were administered via pellets of cells: Noggin-expressing
rat B1 cells (kind gift of R. Harland), or COS cells transiently transfected
with Dkk1 (kind gift of E. Laufer), Crescent (kind gift of M. Marvin and E.
Laufer) or soluble NFz8 (Deardorff et al.,
1998) as described (Streit et
al., 1998; Skromne and Stern,
2001). For combinations of these factors, pellets were made from a
mixture of cells (1000 cells for each factor).

Xenopus experiments

Xenopus oocytes were fertilized in vitro and the embryos staged
according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967).
The cSmad6 (a kind gift from P. Szendro and G. Eichele)
(Yamada et al., 1999) coding
sequence in pCS2+, FGF8 pCS2+ (a kind gift from R. Mayor)
(Christen and Slack, 1997) and
eFGF pCS2+ (Xenopus FGF4, a kind gift from J Slack)
(Lombardo and Slack, 1997)
were used to produce mRNA. Capped mRNA was made with mMessage mMachine
(Ambion). Microinjection was performed as described
(Marchant et al., 1998). The
capped mRNA was injected into the animal zone of two-cell stage embryos, into
the ventral marginal zone of four-cell stage embryos or into blastomere A4 at
the 32-cell stage together with 5-10 ng lysine-fixable fluorescein dextran
(FDX, 40,000 Mr; Molecular Probes) as a lineage tracer in
most experiments. Animal caps were dissected with eyebrow knives at stage 8-10
with the embryos in 0.75× NAM, and were allowed to grow until their
siblings reached stage 21. The accuracy of injection into the A4 blastomere
was assessed by the fate of its progeny. Embryos in which labelled cells were
found in regions other than the ventral epidermis were discarded without
further processing. The proportion of embryos discarded was almost identical
in control (18/51=35% embryos with incorrect labelling) and
Smad6-injected embryos (29/79=37%). In situ hybridization was carried
out as described (Linker et al.,
2000).

Results

Inhibition of BMP signalling by Smad6 is not sufficient for neural
induction

Smad6 is a universal inhibitor of BMP signalling through Smad1/Smad5/Smad8,
without also inhibiting activin/nodal-related signalling through Smad2/Smad3
(Imamura et al., 1997;
Casellas and Brivanlou, 1998;
Hata et al., 1998;
Nakayama et al., 1998;
Bai et al., 2000;
Ishida et al., 2000). We took
advantage of this property to block BMP signalling in the chick embryo. To
confirm that chick Smad6 is active in blocking BMP signalling, we first
electroporated an expression construct
(pCAβ-cSmad6-IRES-GFP) into one side of a stage 3+
embryo and allowed it to grow until the neural tube had formed and closed
(stage 9-10). The embryo was then stained with an antibody against
phospho-Smad1 (Chang et al.,
2002), which revealed that the electroporated side of the neural
tube contained a significantly lower level of activated Smad1
(Fig. 1A-D). As an additional
test, we injected chick Smad6 mRNA (400 pg-1 ng) into the ventral
marginal zone of four-cell stage Xenopus embryos and grew these to
tadpole stages (Fig. 1E). Out
of 207 injected embryos, 196 (95%) formed a secondary axis when compared with
0/72 embryos injected with the same concentration of GFP mRNA
(Fig. 1F,G). These results
confirm that chick Smad6 is active as a BMP inhibitor in both Xenopus
and chick embryos.

Smad6 does not induce neural markers in the chick. (A-G)
Experiments to test the activity of Smad6. Smad6-IRES-GFP was
electroporated into one half of the chick neural tube (A,B). Staining against
phospho-Smad1 (A) reveals that activation of Smad1 has been inhibited in the
electroporated cells (green in B), while in control GFP electroporated embryos
(C,D) phospho-Smad1 is not altered (C). cSmad6 injection into the
marginal zone in Xenopus (E) induces a secondary axis (F), while
GFP-injected controls appear normal (G). (H-N) Smad6 does not induce
neural markers. Electroporation of Smad6 into competent area opaca
epiblast at stage 3+ (H) does not induce Brachyury (I,L; light blue),
Sox2 (J) or Sox3 (M) (purple). In this and subsequent
figures, electroporated cells were visualized by staining with anti-GFP
antibody (K, N; brown). (O-Q) Positive controls. cSmad6 or GFP was
injected at the two-cell stage and animal caps isolated at early gastrula (O).
Smad6 (P) can neuralize animal caps, while GFP cannot (Q), as
assessed by Sox3 expression.

To test whether BMP inhibition by Smad6 is sufficient to cause competent
epiblast cells to acquire expression of early (Sox3) or definitive
neural markers (Sox2), we electroporated
pCAβ-cSmad6-IRES-GFP in a discrete domain in the inner
third of the area opaca of a stage 3+ chick embryo, at approximately the level
of Hensen's node (Fig. 1H).
After incubation for 16-22 hours, no expression of Sox3 (0/7;
Fig. 1I-K), Sox2
(0/16; Fig. 1L-N) or the
mesodermal marker brachyury (0/22;
Fig. 1I,L) was detected in the
electroporated region. As a positive control, we tested whether chick
Smad6 construct can indeed neuralize Xenopus animal caps, as
previously reported (Hata et al.,
1998; Nakayama et al.,
1998). Injection of cSmad6 mRNA into the animal pole of
two-cell stage embryos, followed by culture of their isolated animal caps
leads to strong expression of Sox3 (cSmad6: 40/40;
GFP control: 0/23; Fig.
1O-Q).

Despite published reports that Smad6 should inhibit all BMP signalling
(reviewed by von Bubnoff and Cho,
2001), it is conceivable that some escapes inhibition in our
experimental setup. To overcome this, we misexpressed a dominant-negative form
of the BMP receptor (Suzuki et al.,
1994), Chordin or Noggin, or a combination of all of the above,
with Smad6. In all cases (dnBMPR 0/6; Noggin 0/6; Chordin 0/2;
Smad6+dnBMPR+Noggin+Chordin 0/9), no expression of Sox2 was seen in
the electroporated region (Fig.
2).

BMP inhibition is not sufficient for neural induction. Inhibition of BMP by
misexpression of dnBMPR (A,B), Chordin (C,D), Noggin (G) or
dnBMPR+Chordin+Noggin+Smad6 (E,F) is not sufficient
to induce Sox2. (H,I) Sections through the embryos in F,G at the
levels indicated. All cell pellets produce background staining after
Sox2 in situ hybridization (E-I); sections are therefore necessary to
show absence of expression in the epiblast.

Our results strongly suggest that BMP inhibition, even under conditions
that are likely to abolish all BMP signalling, is not sufficient to induce
expression of either early (Sox3) or definitive (Sox2)
neural markers in competent epiblast cells.

BMP inhibition is required as a late event in formation of the neural
plate

The above experiments suggest that BMP inhibition is not sufficient for
neural induction – but is it necessary? To address this, we
electroporated Xenopus BMP4
(pCAβ-XBMP4-IRESGFP) into the prospective neural plate
of stage 3+ embryos, and analysed the effects in time course. After 12 and 15
hours of incubation, the early marker Sox3 is not affected (0/14 at
12 hours, 0/8 at 15 hours; Fig.
3A,B,G,H), while the later marker Sox2 is strongly
downregulated in the neural plate (9/9 at 12 hours, 10/10 at 15 hours;
Fig. 3C,D,I,J). At 20 hours,
both Sox3 (10/10, Fig.
3M,N) and Sox2 (14/14;
Fig. 3O,P) are downregulated.
At this time, histological analysis showed that neural plate morphology is
lost in the electroporated region (not shown). By contrast, control embryos
electroporated with GFP show no downregulation of either marker at
any time point (0/11; Fig.
3E,F,K,L,Q,R). These results show that BMP inhibition is necessary
for expression of definitive neural plate markers and for neural plate
formation, but does not appear to affect the early steps of this process.

BMP inhibits late markers of neural induction. BMP4 was
electroporated into the prospective neural plate at stage 3+ and the
consequences analysed in time course. After 12 hours and 15 hours of
incubation, the early marker Sox3 is not affected (A,B,G,H), while
the later marker Sox2 is strongly downregulated in the neural plate
(C,D,I,J). By 20 hours after electroporation, both Sox3 (M,N) and
Sox2 (O,P) are downregulated. Neither Sox3 nor Sox2
is altered when control GFP is electroporated (E,F,K,L,Q,R).

BMP inhibition is not sufficient for neural induction even in
combination with FGFs and Wnt antagonists

Recent work has implicated FGF signalling as an early step in neural
induction in the chick (Henrique et al.,
1997; Alvarez et al.,
1998; Storey et al.,
1998; Streit et al.,
2000; Wilson et al.,
2000; Wilson et al.,
2001), in combination with BMP inhibition. One group has proposed
that FGF signalling can cooperate with Wnt antagonism to inhibit BMP activity
and thus induce neural fates (Wilson and
Edlund, 2001; Wilson et al.,
2001). To test this hypothesis, we misexpressed Smad6
with FGFs (FGF2, FGF3, FGF4 or FGF8), with a combination of Wnt antagonists
(Dkk1, Crescent, NFz8 and Cerberus) or all of these together. As shown
previously (Streit et al.,
2000), FGF8 alone induces Sox3 (5/6; 83%) but not
Sox2 (0/8; Fig. 4A) in
the epiblast of the area opaca. FGF8 still fails to induce Sox2 when
misexpressed with Smad6 (0/11;
Fig. 4F,H,I), consistent with
the finding that FGF8+Chordin fail to induce Sox2
(Streit et al., 2000). Similar
results are seen when FGF8 is misexpressed together with three Wnt antagonists
(Dkk1, Crescent and NFz8) (0/7; Fig.
4C-E), or when any of the same Wnt antagonists is misexpressed
individually (Dkk1, 0/9; Crescent, 0/5; NFz8, 0/7;
Fig. 4B,D). More dramatically,
even ectopic expression of FGF8+Smad6+all three Wnt antagonists is unable to
induce Sox2 in competent epiblast (0/10; not shown), and the same is
seen when Cerberus is also included in the combination
(FGF8+Smad6+Dkk1+Crescent+NFz8+ Cerberus: 0/11;
Fig. 4G,J-K). These experiments
show that BMP inhibition is insufficient for neural induction, even in
combination with FGF8 and/or Wnt antagonism.

BMP inhibition does not induce neural tissue even in combination with FGF8
and/or Wnt antagonists. Neither a source of FGF8 protein (A) nor inhibition of
Wnt signalling by NFz8+Dkk+ Crescent (αWnt; pellet of transfected cells)
(B,D) induces Sox2 expression in competent area opaca epiblast. The
same is seen after misexpression of a combination of FGF8+NFz8+Dkk+Crescent
(C,E), FGF8+Smad6 (F,H,I), or of all of these together
(FGF8+NFz8+Dkk+Crescent+Cerberus+Smad6) (G,J,K). The histological
sections (D-G) show that the epiblast in direct apposition to the source of
factors does not express Sox2.

It has been suggested (Wilson et al.,
2000) that FGF3, rather than FGF8, is the endogenous signal, and
these authors' experiments were conducted with FGF2. We therefore tested these
two FGFs as well as FGF4 in the same assays
(Fig. 5). Neither FGF2 (0/7)
nor FGF3 (0/7) alone, nor either factor in combination with Smad6
(FGF2+Smad6: 0/5; FGF3+Smad6: 0/4) or Smad6+Wnt antagonists+Cerberus
(FGF2: 0/6; FGF3: 0/5) induced Sox2
(Fig. 5A-P) at concentrations
of FGF that did not induce brachyury (see Materials and methods). A
more complicated result was obtained with FGF4. At 0.05 μg/ml this factor
alone does not induce brachyury or Sox2 (0/13;
Fig. 5Q,R). However, it induces
both markers when co-expressed with Smad6 (6/6 for
brachyury, 5/6 for Sox2;
Fig. 5S-U). When Wnt
antagonists and Cerberus are misexpressed with FGF4 and Smad6, the induction
of both markers is abolished (0/6; Fig.
5V-X).

BMP inhibition in combination with FGF (FGF2, FGF3 or FGF4) and Wnt
antagonists does not induce neural tissue directly. (A,B,I,J,Q,R) Beads with
FGF2, FGF3 or FGF4 protein cannot induce either brachyury (light
blue) or Sox2 (purple). (C-H,K-P) Likewise, FGF2 or FGF3 together
with Smad6 and/or Wnt antagonists does not induce brachyury
or Sox2. (S-U) Misexpression of Smad6 and FGF4 induces both
brachyury and Sox2; (V-X) addition of Wnt antagonists to the
combination inhibits induction of both markers.

Taken together, these results show that FGF2, FGF3, FGF4 or FGF8 are all
unable to induce Sox2 expression in the absence of mesoderm, even
when any of them is misexpressed together with BMP inhibitors and Wnt
inhibitors.

BMP inhibition by Smad6 is not sufficient for neural induction in
Xenopus

The above results in the chick are in direct conflict with the dominant
`default model' (Hemmati-Brivanlou and
Melton, 1997; Weinstein and
Hemmati-Brivanlou, 1999;
Muñoz-Sanjuán and Brivanlou,
2002) that was based on experiments in Xenopus embryos.
In these experiments, mRNA encoding BMP antagonists is usually injected at the
animal pole of early embryos (one- to four-cell stage), where the RNA may be
inherited not only by prospective epidermal cells but also by presumptive
neural plate or crest cells. To overcome this problem and to generate an assay
more directly comparable with those in chick embryos, we injected a lineage
tracer together with cSmad6 mRNA (400 pg-1 ng) into the A4 (most
ventral animal) blastomere at the 32-cell stage
(Fig. 6A): this is the only
blastomere that does not consistently contribute progeny to neural plate or
neural crest in intact embryos (Dale and
Slack, 1987; Moody,
1987a). Embryos were then grown to the neurula stage and probed
with Sox3 (a definitive neural marker in Xenopus). Both in
Smad6-injected embryos and in GFP-injected controls, normal
expression of Sox3 was seen in the neural plate, but no ectopic
expression was ever seen in the injected cells, which contributed to the most
ventral epidermis (Smad6, 0/26,
Fig. 6B-E; control, 0/21,
Fig. 6F-I). An identical result
was obtained using NCAM as a neural marker (0/5; not shown).

BMP inhibition by Smad6 is not sufficient for neural induction in
Xenopus ventral epidermis. (A-I) cSmad6 was targeted to the
ventral epidermis by injection into the A4 blastomere of 32-cell stage embryos
(A). Smad6 does not activate Sox3 expression (B-E), compare
with GFP-injected control embryos (F-I). B,C,F,G are dorsal views;
D,E,H,I are ventral views of the embryos in B,C,F,G. C,E,G,I show the embryos
in B,D,F,H after staining with anti-fluorescein to reveal the lineage tracer
FDX which was co-injected with the mRNA. (J-L) To test that the progeny of the
A4 blastomere is competent to respond to neural inducing signals from the
organizer, a lineage tracer (FDX) was injected into the A4 blastomere. At late
blastula/early gastrula stage, the labelled cells are transplanted into the
dorsal side of an unlabelled host embryo, which is grown to early neurula
stage (J). (K,L) The transplanted cells (FDX in brown in L) contribute to the
neural plate and express Sox3 (purple).

As differences in competence for mesoderm induction have been reported
along the dorsoventral axis of the early frog embryo
(Sokol and Melton, 1991), it
is conceivable that the A4 blastomere and its descendents are not competent to
respond to neural inducing signals, which might account for the above result.
To test this, we injected a lineage tracer into the A4 blastomere, grew the
embryos to early gastrula stage (stage 10) and then excised the labelled
ventral ectoderm cells and grafted them to the dorsal side (adjacent to the
organizer) of an unlabelled host embryo, which was then grown to neurula
stages and processed for Sox3 and for the lineage marker
(Fig. 5J). The grafted
(labelled) descendants of A4 had become incorporated into the neural plate and
expressed Sox3 normally (n=12;
Fig. 5K,L).

Effects of BMP inhibition in combination with FGF in Xenopus.
Embryos were injected into the A4 blastomere at the 32-cell stage.
FGF4 in combination with cSmad6 does not induce
brachyury in prospective ventral ectoderm cells at stage 10+ (A-C).
Neither FGF8 nor FGF4 is able to induce Sox3 in the
ventral epidermis cells of early neurula stage embryos (D-I), while
misexpression of FGF4 together with Smad6 induces a patch of
Sox3 (J-L). (A,D,G,J) Dorsal views; (B,E,H,K) ventral views of same
embryos, which are shown again in C,F,I,L after anti-fluorescein staining.

These results show that in a blastomere that contributes consistently to
epidermis, BMP inhibition (through misexpression of Smad6) is not sufficient
to cause the injected cells to adopt a neural fate, which argues against the
default model. FGF8 is also not sufficient either alone or in
combination with BMP antagonists to induce Sox3. However, unlike our
findings in chick, the combination of FGF4+Smad6 induces
Sox3 without inducing five different mesodermal markers tested.

Discussion

BMP signalling in neural induction and the default model

The predominant model for neural induction (the `default model') proposes
neural fate as the natural pathway that ectodermal cells would acquire in the
absence of any signal. This fate is inhibited in the ectoderm by active BMP
signalling. The organizer secretes BMP antagonists, which block BMP signalling
in adjacent cells, allowing them to follow their default neural pathway
(Hemmati-Brivanlou and Melton,
1997; Weinstein and
Hemmati-Brivanlou, 1999;
Muñoz-Sanjuán and Brivanlou,
2002). This model is supported by a substantial body of evidence,
mainly from Xenopus embryos. Dissociated animal cap cells that are
subsequently reaggregated develop into neurons
(Godsave and Slack, 1989;
Grunz and Tacke, 1989;
Sato and Sargent, 1989), and
this can be blocked by addition of BMP4 protein
(Wilson and Hemmati-Brivanlou,
1995). The expression patterns of BMP4 and its
antagonists in Xenopus are also consistent with the model:
BMP4 is initially ubiquitous in the ectoderm, and then clears from
the neural plate, while many BMP antagonists (including chordin, noggin,
follistatin, DAN and cerberus) are expressed in the organizer, a
part thereof, or closely neighbouring tissues
(Harland and Gerhart, 1997;
Hemmati-Brivanlou and Melton,
1997; Weinstein and
Hemmati-Brivanlou, 1999;
Muñoz-Sanjuán and Brivanlou,
2002). Misexpression of BMP in the prospective neural plate
ventralizes the embryo, as well as suppressing neural markers, while
misexpression of BMP antagonists (both natural and artificial, such as dnBMPR)
in a one- to four-cell stage embryo expands the neural plate. But most of the
evidence in favour of the model comes from animal cap experiments, where the
early (one- to four-cell stage) embryo is injected animally with an RNA of
choice, then grown to blastula or early gastrula stage, when the animal cap is
cut and allowed to develop in isolation from the rest of the embryo. Caps
obtained from embryos injected with BMP antagonists develop expression of
neural markers, while control caps do not. Animal caps from uninjected embryos
also express neural markers if they are treated with any of the endogenous BMP
antagonists (Noggin, Chordin, Follistatin, Cerberus, etc.) in protein form
(Hemmati-Brivanlou et al.,
1994; Hemmati-Brivanlou and
Melton, 1994; Sasai et al.,
1995; Bouwmeester et al.,
1996).

Although several objections have been raised to the model and to the
interpretation of experiments that led to it (see
Streit and Stern, 1999c), this
model is generally so dominant that it is described in all current
developmental biology textbooks as the accepted mechanism for neural
induction.

Conflicting data from chick and other species

The first major objections to the default model as a sufficient explanation
for neural induction were raised as a result of observations in the chick
(Streit et al., 1998): BMP4,
BMP7 and their antagonists Chordin, Noggin and Follistatin are not expressed
with the correct spatial and temporal patterns to fit neatly with the
proposals of the model, misexpression of Chordin or Noggin in competent
epiblast using grafts of secreting cells do not induce neural tissue, and
misexpression of BMP4 or BMP7 by the same method in the prospective neural
plate does not block neural induction. Furthermore cell dissociation of chick
epiblast does not induce neural differentiation but rather muscle
(George-Weinstein et al.,
1996; George-Weinstein et al.,
1997). Consistent with these results in the chick, mouse mutants
that lack Chordin, Noggin or both BMP antagonists still have a nervous system,
although they lack the most anterior structures
(McMahon et al., 1998;
Bachiller et al., 2000;
Belo et al., 2000;
Mukhopadhyay et al.,
2001).

However, chick epiblast cells previously exposed to a grafted organizer for
at least 5 hours (13 hours are required for full induction)
(Gallera and Ivanov, 1964;
Gallera, 1971) can respond to
Chordin by stabilizing the expression of the early marker Sox3 (but
still do not express the definitive neural marker Sox2)
(Streit et al., 1998;
Streit et al., 2000). This
finding led to the hypothesis that signals other than BMP antagonists are
required to confer sensitivity to epiblast cells to BMP signalling, and
therefore that BMP inhibition may be a relatively downstream step in the
induction process, if it is required at all.

But these experiments are open to three major criticisms. First, they
cannot address whether it is necessary to inhibit BMP signalling at all for
neural induction to occur. Second, it is possible that misexpression of BMP or
its antagonists using a graft of secreting cells does not deliver enough
active protein to overcome the endogenous signals. Third, it is possible that
any one of the BMP antagonists is not sufficient to inhibit all BMP
signalling.

Here we have addressed the first question using electroporation of BMP4 in
an expression construct directly into the epiblast; we find that BMP
inhibition is indeed necessary for neural plate development. However, although
misexpression of BMP4 affects the definitive neural marker Sox2, it
does not alter the expression of the early marker Sox3 [which at
early stages is not restricted to prospective neural cells but is also
expressed in future epidermis and mesoderm cells (see
Sheng et al., 2003)],
consistent with the idea that BMP inhibition is a relatively late step in a
cascade leading to neural induction in the chick.

To address the latter two criticisms, we electroporated the cell-autonomous
BMP antagonist Smad6, either alone or together with other BMP antagonists
(Chordin, Noggin and/or dnBMPR). In none of these cases do
we see induction of either Sox3 or Sox2 in competent
epiblast of the area opaca, now strongly suggesting that BMP inhibition is not
sufficient for neural induction in the chick.

FGF signalling and neural induction

The finding (see above) that signals from the organizer other than BMP
antagonists are required as an upstream step before chick epiblast cells can
respond to the antagonists led to a screen for genes that are activated during
the initial signalling period. To date, two genes have been described from
this screen: ERNI (which is induced in just 1 hour) and
Churchill (induced after 4-5 hours)
(Streit et al., 2000;
Sheng et al., 2003). In turn,
the use of ERNI as a marker led to the identification of FGF
signalling as both necessary and sufficient to induce all the known early
markers (ERNI, Sox3 and Churchill) as well as to sensitize
cells to Chordin; however, it is not sufficient to induce Sox2 or a
neural plate (Streit et al.,
2000; Sheng et al.,
2003). It was also shown that this early FGF step takes place
during very early stages of development, even before gastrulation begins
(Streit et al., 2000;
Wilson et al., 2000).

Recently, it has been shown that signalling by FGF and by other secreted
proteins that work through MAP kinase act in part by phosphorylating the
linker region of Smad1, rather than the C terminus as does BMP signalling
(Pera et al., 2003). A
double-phosphorylation mechanism was therefore proposed as a molecular basis
for the cooperation between FGF and BMP in neural induction and other
embryonic signalling events (Pera et al.,
2003). However, a more recent study in mouse by Soriano and
colleagues has elegantly demonstrated that mice carrying mutations in these
two distinct domains of Smad1 show different and additive phenotypes, and that
the mutations cannot complement each other, suggesting that linker and
C-terminal phosphorylation of Smad1 (and thus MAPK and BMP signalling) have
different functions during early development
(Aubin et al., 2004).

Our present experiments demonstrate that in the chick, neither FGF2, FGF3,
FGF4 nor FGF8 is a sufficient neural inducer in the absence of
brachyury expression, even when any of these is combined with Smad6
as a BMP antagonist, consistent with the previous findings that FGF8+Chordin
do not induce Sox2 expression when administered as proteins
(Streit et al., 2000). These
results suggest that FGFs may only be able to induce definitive neural tissue
in cooperation with other signals in addition to BMP antagonists.

A role for Wnt signalling in neural induction?

More recent experiments in the chick, using NFz8 as the Wnt antagonist,
explant assays from the area pellucida and an antibody against Sox2, suggested
that Wnt inhibition together with FGF can act as a sufficient neural inducer,
and FGF3 was suggested as the endogenous factor
(Wilson et al., 2001). These
experiments were interpreted as indicating that BMP signalling can be
inhibited by these treatments through an alternative pathway and that the key
event may be downregulation of BMP at a transcriptional level
(Bainter et al., 2001;
Wilson and Edlund, 2001). In
Xenopus, however, Wnt signalling seems to promote neural induction
(Baker et al., 1999), although
it is thought that the conflict is resolved by differential timing of these
events: Wnt signalling is required in early (pre-gastrula) stages of
development, while inhibition of Wnt may be important for acquisition of
neural fate at later stages (Bainter et
al., 2001; Wilson and Edlund,
2001).

To test whether inhibition of Wnt signalling can cooperate with BMP
antagonism and/or FGF signalling in vivo, we misexpressed combinations of
these agents in competent epiblast. Even a combination of FGF (FGF2, FGF3,
FGF4 or FGF8), Smad6 and four different Wnt antagonists (alone or in
combination) is unable to induce Sox2 expression in this assay. The
difference between our result and those obtained by Wilson et al.
(Wilson et al., 2001) are
difficult to explain, but it is possible either that isolated explants
cultured for 48 hours are somehow sensitized to neural inducing signals, or
that the Sox2 antibody might crossreact with Sox3 (which is induced by FGF8).
We have attempted to use this same antibody for our experiments with a variety
of protocols, but were unable to obtain reliable background-free staining
(data not shown). Based on the results of the present experiments, we can only
conclude that even a combination of FGF+Smad6+anti-Wnt is insufficient to
mimic the effects of a grafted organizer and induce Sox2 expression,
or an ectopic neural plate, in competent epiblast of the area opaca. We
suggest that neural induction is a multi-step process that begins very early
in development and involves other neural inducing factors, which remain to be
identified.

Do different vertebrates use different mechanisms to specify neural
fate?

The results of our present experiments in the chick, along with those
previously published by ourselves and other groups, still raise the issue of
whether different vertebrates might use different molecular pathways to induce
the nervous system. We therefore tested whether inhibition of BMP signalling
with Smad6 is sufficient for neural induction in Xenopus.

Most experiments on neural induction in Xenopus have been
conducted on animal caps cut from embryos injected at the animal pole at the
one- to four-cell stage. It is important to bear in mind that such animal caps
almost certainly include some prospective neural plate or neural crest cells
(Jacobson and Hirose, 1981;
Dale and Slack, 1987;
Moody, 1987a;
Moody, 1987b;
Wetts and Fraser, 1989;
Eagleson and Harris, 1990;
Saint-Jeannet and Dawid, 1994;
Delarue et al., 1997). It is
conceivable that prior to their isolation from the embryo, these cells have
received some signals that, although not sufficient for neural induction, may
represent some early steps in the process. For this reason, we chose to target
Smad6 to the most ventral animal blastomere at the 32-cell stage, as this is
the only blastomere that does not consistently contribute to neural plate or
neural crest, but mainly to the most ventral (belly) epidermis. Using a
concentration of Smad6 mRNA that is sufficient to induce axial
duplications when targeted to the marginal zone (400 pg to 1 ng), and also
sufficient to induce neural tissue in animal caps (1 ng), we see no ectopic
expression of the neural markers Sox3 (which in Xenopus is a
definitive neural plate marker) or NCAM in the descendants of the
injected A4 cell. These findings suggest that, in the Xenopus embryo
as well as in the chick (Streit et al.,
1998; Streit and Stern,
1999a; Streit and Stern,
1999b), BMP inhibition may only be sufficient to deviate the
border of the neural plate when the antagonists are targeted to its vicinity,
but is insufficient to cause prospective epidermis (cells fated for neither
the neural plate nor its border) to acquire neural traits. Therefore animal
cap assays are not a good test for whether a candidate molecule has neural
inducing activity.

Our chick and Xenopus results differ in one respect. Misexpression
of FGF4 (at concentrations that do not induce expression of
brachyury) with Smad6 induces Sox3 in the absence
of mesoderm markers in Xenopus, but does not elicit a comparable
response (Sox2) in chick. There are several possible interpretations
for this difference. (1) There may be real differences in the mechanism of
neural induction in the two species. We feel that this is unlikely as
cross-species grafts of the organizer work very well across all vertebrate
classes (Waddington, 1934;
Waddington, 1936;
Waddington, 1937;
Kintner and Dodd, 1991;
Blum et al., 1992;
Hatta and Takahashi, 1996).
(2) The expression patterns of FGF4/eFGF are different in the two species–
in chick FGF4 is expressed in mid-posterior streak but not in
the organizer (Streit and Stern,
1999b), while in Xenopus, eFGF is expressed in a domain
including the dorsal lip (Isaacs et al.,
1992; Isaacs et al.,
1995); different FGFs may therefore fulfill this function in the
two species. (3) It is possible that either endoderm or lateral mesoderm
(which do not express any of the five markers tested) is induced in
Xenopus. (4) It is possible that the level of inductive signal
provided by Smad6+FGF4 is not sufficient to induce mesoderm, but enough to
cause injected cells to produce some products normally secreted by early
prospective mesendoderm cells. (5) It is possible that the
brachyury-expressing cells in the chick are prospective
caudal neural plate, which has been shown to express this marker in
chick (Storey et al., 1998)
but not in Xenopus. Although this explanation seems the most likely,
we have observed that the induced Sox2 expression is restricted to
the Smad6-electroporated cells (marked by GFP), while brachyury is
also expressed in neighbouring cells; the possibility therefore remains that
the Sox2 induction in this experiment is indirect. Furthermore, this
does not explain the finding that in the presence of Wnt antagonists both
brachyury and Sox2 expression disappear. Finally, (6) in
Xenopus, the marker selected for assessing neural plate is
Sox3, while in chick the definitive marker is Sox2. These
genes (and the closely related Sox1) appear partly to have swapped
functions in evolution, even between birds and mammals
(Uwanogho et al., 1995;
Collignon et al., 1996); it is
therefore very likely that the enhancer elements regulating the expression of
these two markers differ in the two species. Furthermore, Sox3 is not
an exclusive neural marker in any of the vertebrate classes, and the induced
expression seen in Xenopus could correspond to a different cell
type.

Conclusion: neural induction as a multi-step process

Our experiments provide evidence that BMP inhibition is required for neural
induction in the chick, but only as a relatively late step in a molecular
cascade. They also strongly suggest that BMP inhibition is not sufficient to
cause competent ectodermal cells to acquire neural fates either in chick or in
Xenopus. In Xenopus, FGF synergizes with BMP inhibition to
induce neural markers (we cannot yet conclude definitively whether this
combination is sufficient). In chick, inhibition of BMP signalling, even
together with Wnt antagonists and/or FGF, is not sufficient for neural
induction. We propose that neural induction does not occur `by default' but
rather that it involves a succession of signalling events, where some players
remain to be identified.

Acknowledgments

We are indebted to Ali Hemmati-Brivanlou who first suggested to us the use
of Smad6 to test the default model, which led to this study; and to Irene De
Almeida for conducting a first set of experiments with Smad7 and Wnt
antagonists. We are also grateful to Laurent Kodjabachian and Patrick Lemaire
for sharing data prior to publication and for insightful discussions; and to
Leslie Dale, Roberto Mayor and Andrea Streit for useful comments on the
manuscript and advice. We also thank Christelle Devader, Tim Geach, Leslie
Dale, Claudio Araya, Jaime De Calisto and Roberto Mayor for help with
Xenopus manipulations; Sharon Boast and Mario dos Reis for technical
support; and Costis Papanayotou, Federica Bertocchini, Guojun Sheng and
Octavian Voiculescu for useful discussions. This study was funded by grants
from NIMH (5R01MH060156), the MRC and the Wellcome Trust. C.L. was funded by
an EMBO long-term fellowship.

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